Nature - USA (2020-01-16)

Nature | Vol 577 | 16 January 2020 | 393

analysis, and comparison to scRNA-seq data from adult trigeminal
ganglia (see Methods, Extended Data Figs. 2a, b, 3a–d, Supplementary
Data 1). These cell-type classifications are consistent with previously
published RNA-seq findings of adult DRG and trigeminal ganglia^16 –^19.
We next sought to determine how the transcriptional identities of
mature somatosensory neuron subtypes compare to those of newborn
sensory neurons by analysing the transcriptomes of cells from DRG at
E11.5 (Fig. 1b). We visualized the E11.5 scRNA-seq data using uniform
manifold approximation and projection (UMAP)^20. Mapping historically
defined marker genes onto the UMAP representation revealed three
principal cell types in E11.5 ganglia: 1) multipotent neural crest progeni-
tors (NCPs), marked by Sox10^21 ,^22 ; 2) sensory neuron progenitors (SNPs),
marked by Neurog1 and Neurog2^23 ; and 3) nascent, postmitotic sensory
neurons marked by expression of the somatosensory neuron gene advil-
lin (Avil^24 ) and concomitant loss of expression of cell-cycle-associated
genes (Fig. 1b, Extended Data Fig. 4a). Monocle 3^25 was then used to infer

developmental relationships between NCPs, SNPs, and nascent Avil+

sensory neurons. This analysis revealed a single continuous trajectory

connecting NCPs, SNPs, and Avil+ sensory neurons, suggesting a lack

of transcriptional diversity in sensory neuron progenitors populating

the Avil+ compartment. Unexpectedly, labelled descendants of Neu-

rog1+ SNPs from Neurog1Cre; Rosa26LSL-tdTomato mice had a broad range

of cell diameters (Fig. 1c), consistent with the previous finding that

in vitro directed differentiation with Neurog1 overexpression leads to

the generation of both small- and large-diameter DRG sensory neuron

subtypes^26. Moreover, genes that were highly enriched in E11.5 Avil+

sensory neurons, relative to progenitors, generally remained expressed

in adult somatosensory neuron subtypes (Extended Data Fig. 4b). On

the other hand, the majority of genes whose expression was restricted

to individual terminal somatosensory subtypes of adult ganglia were

expressed at trace levels in E11.5 Avil+ sensory neurons (Fig. 1d), sug-

gesting that upon cell-cycle exit sensory neurons are transcriptionally

Postnatal day 5

SST

MRGPRD+

Proprioceptors

Aβ RA-LTMR

Aβ eld/SA1

C-LTMR

CGRP-α

CGRP-ε

CGRP-η

CGRP-γ

CGRP-θ

CGRP-ζ

Cold thermoceptors

Unassigned/

non-neuronal

Aδ-LTMR

SST

CGRP-θ

Aδ-LTMR

C-LT MR

Aβ RA

• LTM
  R

Proprioceptors

CGRP-α

CGRP-γCGRP-η

CGRP-CGRP-ζ ε

Aβ

eld/SA1

Cold

thermoceptors

MRGPRD+ polymodal

nociceptors

Cold

thermoceptors

CGRP-αCGRP-γ

CGRP-η

C-LTMR

MRGPRD

+ polymodal

nociceptors

SST

CGRP-θCGRP-ε

CGRP-ζ

Aβ eld/SA1

Aδ-LTMR

Aβ RA-LTMR

Proprioceptors

Propr

ioceptors

Aδ-L

TMR

C-LTMRMRGPRD

+ thermo.Cold

CGRP-CGRP-α

θ

CGRP-

ε

CGRP-

ζ

Aβ RA-L

TMR

Aβ eld/SA1

CGRP-η

Adult Postnatal day 0

Embryonic day 15.5 Embryonic day 12.5

a

Neural crest progenitor

Unspecialized sensory neuron

Neural crest progenitors

(Sox10)

0

2

Unspecialized sensory neurons

(Avil)

0

1

ln(TPT+1)

Embryonic day 11.5

ln(TPT+1)

Sensory neuron progenitors

(Neurog1)

0

3

ln(TPT+1)

Sensory neuron progenitor

Infer

red trajectory

Inferred trajectory tdTomato tdTNeuNomato^75 μm

Neurog1Cre/WT;Rosa26LSL-tdTomato/WT

100

tdT 0

omato

+

neur

ons (%)

<20

μm

20–30

μm

>30

μm

b c

d

Aβ eld/SA1

Aβ (^) RA-L
TMR
Aδ-LTMR
CGRP-α C-LTMR
CGRP-ε CGRP-η
CGRP-θ CGRP-γ
CGRP-ζ
MRGPRD+ polymodal
nociceptors
Proprioceptors
SST+
Cold
thermoceptors
Non-neuronal
SST
Aβ RA-LTMR
MRGPRD+ polymodal
nociceptors
C-LT MR
CGRP-θ
CGRP-γCGRP-ε
CGRP-ζ
Aδ-LTMR
Aβ eld/SA1Proprioceptors
CGRP-η
Cold
thermoceptors
CGRP-α
Adult
Aβ eld/SA1Aβ RA-LTMR
Adult
Aδ-LTMR
Adult
C-LTMR
Adult Adult
0.5 0.6
CGRP-α CGRP-ε CGRP-η
CGRP-γ CGRP-θ CGRP-ζ MRGPRD+ Proprioceptors SST Cold thermo.
0.4 0.6

---

0.6 0.4 0.6

• 0.6


• 
  

  0.4 0.6

  
  


• 
  

  0.6 0.8

  
  


• 
  
  
  
  • 0.8 0.6
    ln(TPT+1) *
    Cell-type-
    specic genes
    Cells
    ln(TPT+1)
    0 0 0 0 0 0 0
    0 0 0 0 0 0 0
    Adult Adult
    0
    2
    ln(TPT+1)
    USNUSN USN USNUSN USN USN
    USNUAdult SNAdultUSNUAdult SNAdultUSNAdult USNUAdult SNAdult
  
  
  
  


• 
  
  
  
  • 
    
    
    
    • 
      
    
    
    
    
  
  
  
  


• 
  

  NCP
  Neuron
  (cell state)
  Neurog1(Genetic strategy)Cre/WTSNP
  ;Rosa26
  LSL-tdTomato
  /WT
  Fig. 1 | scRNA-seq of developing and mature DRG sensory neurons. a, t-SNE
  visualizations of DRG scRNA-seq data. SST, somatostatin. b, UMAP
  visualization of DRG scRNA-seq data from E11.5 with developmental trajectory
  and gene expression information overlaid. TPT, tags per ten thousand.
  c, Quantification (mean ± s.e.m.) of tdTomato+ neurons and representative
  image (right). d, Heat map and quantification of genes enriched in each
  somatosensory neuron subtype as well as their expression levels in
  unspecialized sensory neurons (USNs). Boxes represent interquartile range
  (IQR), whiskers represent minimum and maximum values, and notches
  represent the 95% confidence interval of the median. *P < 0.0001, two-sided
  Wilcoxon rank-sum test with Bonferroni correction. For n values, see Methods.